The pathogenic event common to all forms of Alzheimer's disease (AD) is the abnormal accumulation of the amyloid 2-peptide (A2) in specific regions of the brain. This accumulation of A2 is considered a key pathological event in AD and is the basis of the so-called amyloid hypothesis of the disease. Therefore, therapeutic approaches that reduce the accumulation of A2 are currently being sought. It has been shown definitively that the generation and clearance of A2 in the CNS is regulated by cholesterol homeostasis. Compounds that perturb cellular free cholesterol homeostasis such as acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors have been shown both in vitro and in vivo to reduce A2 production and secretion. However, it is generally the case that ACAY inhibitors exhibit poor oral activity. The beauveriolides are a new class of fungal metabolites that have been shown to be orally active ACAT inhibitors that are currently being investigated as potential therapeutics for atherosclerosis. NB No studies have been reported to date on the investigation of the A2-inhibitory effect of the natural product beauveriolides or beauveriolide-inspired compounds. The hypothesis being tested in this R21 is that the natural product beauveriolides and libraries of beauveriolide-inspired compounds should reduce A2- production and secretion in vitro via inhibition of ACAT and perturbation of cholesterol homeostasis. They would in such a case be a NEW CLASS of potential AD therapeutics working via an established mechanism of action and hence this proposal is entirely within the remit of the PA-06-261 titled Grants for Alzheimer's Disease Drug Discovery (R21). We will investigate this hypothesis under the remit of the following two specific aims: Specific aim #1. Synthesis of beauveriolide-derived cyclodepsipeptide and cyclopeptide libraries In this specific aim libraries of beauveriolide-inspired structures are proposed (L1/L1', L2, L3 and L4). The key structural aspects being optimized are the macrolactone ring, the lipid side chain and the three hydrophobic amino acid components of the ring. Prior to selection for synthesis, all virtual library members will first be interrogated using in silico methods (the QikProp v3.0 suite of Maestro v 8.0) for favorable properties including Lipinkski's rule of 5, log BB and oral absorbtion. Only those members that fulfill the criterion will be synthesized. The synthesis is proposed to be a mixed solid- phase/solution phase approach. Library synthesis and optimization is proposed to occur in two stages, the first being randomization of the macrolactone and lipid side chain (libraries L1/L1') which will then be screened for biological activity as outlined in specific aim #2. The active compounds from L1/L1'will then be further refined through structural modifications of the hydrophobic amino-acids (libraries L2, L3 and L4) using positional scanning. Specific aim #2: To determine whether synthetic beauveriolide-inspired libraries reduce the levels of 2-secretase cleavage products of APP, including A2 in vitro In this specific aim there are three sub-aims each being a cell-culture assay designed to assess the ability of the library members synthesized in specific aim #1 to either 1. reduce lipid loading or 2. reduce A2 production and secretion. All analyses will be correlated with measurements of total protein production (BCA analysis) and free and esterified cholesterol (Amplex Red assay, Molecular Probes). Sub-aim 2.1 Measuring lipid loading in culture macrophages. Lipid droplet formation will be measured in a cultured murine macrophage cell line (J774.1) that has been treated with cholesterol-loaded liposomes. Quantification of lipid loading will be performed by microscopy of fixed and stained (H&E and oil-red O) cells. Sub-aim 2.2 ELISA assay to measure A2 secretion in cell-culture For this specific aim the CHO cell-lines stably transfected with human APP751 (7WD10 cell line and AC29, gifts from Prof. Ta-Yuan Chang) will be treated with library members from specific aim #1. The levels of A240 and A242 -secreted will be determined using a commercially-available ELISA kit (The Genetics Company, Switzerland). Sub-aim 2.3 Measurement of APP 2-cleavage product (2-APP-CTF) in cell culture In order to test the response of human neuronal cells to the chemical libraries synthesized in specific aim # 1 we will analyze the levels of 2-APP-CTF generated by the SH-SY5Y neuroblastoma cell line (ATCC, VA, US) using Western blot analysis with commercially available antibodies. These cells do not generate a detectable level of A2, but do generate detectable levels of 2-C-terminal fragments (2-APP-CTF), which have been analyzed in previous studies of ACAT inhibition and are indicative of 2-cleavage products in general, one of which is A2. NB CP-113,818 will be studied as a control ACAT inhibitor in this R21 proposal studies (gift from Pfizer Inc., c/o Mr.D. W. Owens, Pfizer Compound transfer manager). PUBLIC HEALTH RELEVANCE: Alzheimer's disease is now the most common form of dementia, affecting up to 15 million people worldwide. As a result of the increase in life expectancy, it is anticipated that by 2050 approximately 25 % of the world population will be over 65, of which one third are likely to develop AD. AD is a complex and genetically heterogeneous disease, characterized by progressive memory deficit, cognitive impairment and personality changes accompanied by specific structural abnormalities in the brain. In monetary terms, AD is already America's third most expensive disease - costing over $100 billion per year. Add to this the social, psychological and emotional costs of sufferers'and carers'diminishing quality of life and it becomes clear that inhibiting the development of Alzheimer's would have a hugely beneficial impact on public health and wealth. This project describes the development of a new class of compounds that may prevent and/or AD.